Bacterial type I modular polyketide synthases (PKSs) are complex, multifunctional enzymes that synthesize structurally diverse and medicinally relevant natural products. Given their modular organization, the manipulation of type I PKSs holds tremendous promise for the generation of novel macrolide antibiotics that are not easily accessible by standard chemical synthetic approaches. To fully harness this potential, however, a thorough evaluation of a variety of PKS systems is needed. To this end, the experiments described within this proposal seek to interface chemical synthesis with rigorous kinetic analysis and structural biology in an effort to enhance our understanding of specificity and catalysis of the pikromycin PKS system. In addition to providing detailed information regarding the kinetics of individual PKS modules, this work will also begin to uncover substrate specificity differences between the pikromycin and erythromycin (DEBS) PKS systems. Furthermore, a detailed understanding of the specificity of the pikromycin thioesterase domain will be achieved via x-ray co-crystallization studies. [unreadable] [unreadable] [unreadable]